Our Research is focused on studies of the safety, immunogenicity/activity and mechanism of action of DNA vaccines and immunomodulatory CpG oligonucleotides (ODN). I. SAFETY STUDIES. Our safety studies examine the ability of DNA vaccines and CpG ODN to a) alter the immune milieu of the host, thereby effecting ongoing and subsequent immune responses; b) to migrate from the site of injection and persist for prolonged periods in vivo (with the potential for integration); c) induce intolerance rather than immunity following DNA vaccination (thereby increasing the host's susceptibility to infection) and d) potentiate the development of autoimmune response: II. STUDIES OF IMMUNOGENICITY/ACTIVITY. DNA vaccines and CpG ODN represent cutting edge technologies that are being harnessed for a variety of therapeutic purposes. Our lab studies a) the ability of DNA vaccines and CpG motifs to induce strong immune responses in mice, monkeys and humans, b) the ability of CpG ODN to act as anti-allergens, down-regulating the production of allergen specific IL-4 and IgE by promoting the development of non-pathologic Th1 responses to allergens and c) the capacity of CpG ODN to up-regulate the innate immune system, thereby enabling to host to resist infection by a variety of pathogenic microorganisms. Finally, we examine the ability of a "suppressive" sequence motifs to block the immune activation induced by CpG ODN, and their ability to inhibit the development of autoimmune diseases. III. STUDIES OF MECHANISM OF ACTION. To understand and improve the therapeutic applications of CpG motifs, we are studying a) the gene activated by CpG and suppressive motifs, b) the intracellular pathways activated by these ODN, and e) the type of cells, and cellular cascades stimulated by their in vivo administration. Immune Activation Induced by viruses and protein-based vaccines. Our lab studies the phenotype, location and functional properties of lymphocytes stimulated to produce antibodies and cytokines when challenged with vaccines, proteins, and DNA derived from bacteria or viruses. This work examines the immunogenicity of the envelope glycoprotein of HIV and the protective antigen from anthrax. Also studied is the effect of adjuvants on the activation of cytokine producing cells, and whether early changes in the cytokine milieu effect subsequent immune responses. In some experiments, animals are challenged with infectious pathogens, and protection following vaccination monitored. This work established that CpG motifs can improve vaccine immunogenicity. We are in the process of establishing the ability of CpG ODN to boost both the titer and avidity of the host's response to anthrax following AVA or rPA immunization. Studies of autoimmunity and tolerance induced by vaccines, hormones, cytokines and retroviruses. A variety of immunostimulatory agents can have unexpected adverse consequences, such as the development of autoimmune disease or the inmduction of tolerance. We examined the mechanisms responsible for the induction of these adverse consequences. Studies were designed to examine the immunostimulatory cascade initiated by protein antigens (including vaccines) and immunostimulatory CpG motifs present in DNA vaccines on the immune milieu, and whether they predisposed to the development of autoimmunity or tolerance. Our experiments show that agents capable of altering the balance between Th1 and Th2 cytokines profoundly impact the initiation, severity and persistence of both systemic and organ specific autoimmunity. Most recently, we confirmed the ability of CpG oligonucleotides to increase susceptibility to inflammatory arthritis, and of suppressive ODN to prevent/treat such arthritic conditions. This provides insight into the role of CpG-containing bacterial DNA to the pathogenesis of autoimmune disease, and raises the possibility of such diseases being treated using suppressive ODN. Safety, immunogenicity and mechanism of action of DNA vaccines. DNA vaccines provide an important new approach to vaccine development. Constructed by introducing antigen-encoding gene segments into a self-replicating plasmid of bacterial DNA, these vaccines are easy to manufacture, purify and administer. We are analyzing the nate and location of immune cells activated by DNA vaccination and their safety and mechanism of action. We are also exploring methods of improving DNA vaccine immunogenicity. This includes incorporating additional CpG motifs, use of lipid vehicles and gene guns for delivery, and by co-administering cytokine-encoding plasmids with the vaccine. We've also explored the ability of DNA plasmids to act as gene therapy agents. We've shown that a plasmid encoding erythropoietin can be introduced by gene gun into the skin of normal mice, and stimulate a significant increase in serum Epo levels and hematocrit. The magnitude and duration of this effect could be regulated by the dose and frequency of plasmid administration. Since transfected skin cells are rapidly shed, safety concerns associated with the long-term transfection of cells are alleviated by this approach. Safety and Activity of Immunostimulatory and Immunosuppressive DNA motifs. We established that oligodeoxynucleotides expressing "CpG" motifs (CpG ODN)mediate a variety of immunological functions, including the activation of B lymphocytes and the induction of Th1 cytokine production by NK, T and dendritic cells. We recently established that "D" type ODN could support the maturation of human peripheral blood monocytes into antigen presenting dendritic cells. We have been exploring the mechanism(s) underlying this immune activation, and found that a 6 base pair DNA motif (containing an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines) that is common to bacterial but not mammalian DNA caused this stimulation in mice. In humans, we found that different motifs (and different types of nucleotide backbones) were required to induce immune activation. We've identified two broad and distinct categories of ODN ("D" and "K" type) that induce different immune responses on human PBMC in vitro and in rhesus macagues in vivo. This induction of different types of immune response may prove useful in optimizing the human response specific pathogens. We found that synthetic oligonucleotides expressing CpG motifs can act as anti-allergens (by deviating the immune response towards Th1 and away from Th2 driven IgE secretion), and can be used to prevent and/or treat bacterial, parasitic and viral infections by up-regulating the innate immune system. We are particularly interested in the ability of CpG ODN to prevent/treat diseases caused by potential biowarfare agents, including Ebola virus and anthrax. We are therefore examining methods of increasing and prolonging the protective effects of CpG ODN. Most recently, we demonstrated that CpG ODN were able to provide some protection against infection by anthrax and Ebola virus in murine models. We also showed that we could significantly reduce the level of Leishmania infection in rhesus monkeys treated with CpG ODN expected to be active in humans. By analyzing the response of >100 normal donors to a large panel of ODN, we found that individuals varied in their response to specific ODN, such that optimal stimulation of a diverse population will require combinations of different ODN motifs. We've also shown that PBMC from patients infected with HIV continue to benefit from the stimulatory activity of CpG ODN. We've also been exploring the role of "suppressive motifs" present in mammalian DNA, and their ability to regulate the innate immune response elicited by CpG motifs. These suppressive motifs may play a critical role insuring that over-exhuberant responses to CpG ODN do not harm the host. This project incorporates FY2002 projects 1Z01BK003005-10, 1Z01BK003015-06, 1Z01BK003016-06, and 1Z01BK003018-04.